Our long-term goal is to determine how gonadotropins function. Gonadotropins require N-glycans to fully activate their cognate receptors even though high affinity binding involves only protein-protein interactions. Gonadotropins are used to treat infertility, but become progressively less effective as patients age. Urinary and recombinant hFSH preparations lack a novel, highly active glycoform, di-glycosylated hFSH, that possesses only a subunit N-glycans and which predominates in pituitaries of young women. The relative abundance of di-glycosylated and tetra-glycosylated hFSH appear to be physiologically regulated. This proposal will define these changes and study possible mechanisms to explain differences in activity via 2 specific aims. 1. Characterize cyclic- and age-related changes in hFSH glycoform abundance. First, we will measure urinary FSH glycoform abundance in young healthy cycling women. Our working hypothesis is that ovarian feedback will alter FSH glycoform abundance. Second, we will evaluate glycoform abundance during the perimenopausal period, as the timing of the shift in glycoform abundance is unknown. One hypothesis is that FSH glycoform abundance changes gradually during the perimenopausal period, thereby contributing to declining fertility. An alternative hypothesis is that glycoform abundance changes abruptly at the menopausal transition. Finally, we will examine glycoform abundance in post-menopausal women and compare individuals receiving estrogen replacement therapy with those receiving none. Our working hypothesis is estrogen selectively inhibits hFSHfi glycosylation. 2. Identify a and /? subunit glycosylation patterns modulating FSH receptor-binding and FSH-stimulated steroidogenesis. We will compare receptor-binding and steroidogenic activities of di- and tetra-glycosylated hFSH. Our hypothesis is that di-glycosylated hFSH on rate is faster than tetra-glycosylated hFSH. The relationship of FSHR binding and steroidogenic activity will be compared. Our working hypothesis is that di-glycosylated hFSH will exhibit greater biological activity than predicted by its significatly higher receptor-binding activity. This project is centered around glycoforms readily identified by western blotting and lays the ground for future studies by contributing to the development of methods to rapidly characterize FSH glycosylation. A perimenopausal marker and more effective preparations for treating infertility are potential practical outcomes of this project.